It is unclear where within brain neurons (which organelles) the amyloid- peptide (Ax) is predominantly produced in Alzheimer's disease (AD) & whether or not the overproduction of Ax that occurs is due to a mistrafficking/misrouting of the amyloid precursor protein (APP) &/or the -secretase enzyme that processes APP in AD. In AD, there is cerebral acidosis (e.g., pH 6.6) & a decreased intracellular pH.1 Since -secretase has an acidic pH optimum, it is important to determine if the intracellular acidosis also leads to decrease in the pH of organelles within which APP is processed. The Aims of the proposed work are to (1) gain a molecular level understanding from computer simulations of how changes in the lumenal pH of crude organelle models (liposomes) impact the A42 self-aggregation & A42-lipid interactions, to (2) establish the baseline pH distribution of the early & late endosome (LE), lysosome (LY), & Golgi apparatus (the organelles within which Ax is potentially produced) within embryonic & adult mouse brain neurons as a function of (a) each organelle- type's proximity to the cell body, to (3) characterize how these distributions change in a transgenic mouse model (5XFAD) of familial Alzheimer's disease (FAD) as a function of (b) the age of the mice & (c) gross brain region (hippocampus & isocortex) when compared to controls, & to (4) characterize how the colocalization of APP, Ax, & -secretase changes within these organelles with respect to a, b, & c. Aim (1) will be achieved using coarse-grained molecular dynamics simulations of liposomes with encapsulated Ax. Aims (2)-(4) will be achieved using fluorescence microscopy techniques. The proposed work is driven by the following Hypotheses: (1) a decreased pH will promote self-aggregation of Ax & interactions of Ax with the liposome walls, leading to disruption of the integrity of the liposome. (2) The pH distribution of neuronal organelles in all non-AD cases will exhibit a spatial gradient in which the organelles proximal to the cell body will be more acidic than distal organelles. (3) As the 5XFAD mice age, there will be (i) acidosis of the Golgi & early endosomes (EEs) & (ii) a statistically significant increase in th colocalization of Ax & the -secretase enzyme in the Golgi & EEs as compared to the LEs & LYs in the AD model. Changes (i) & (ii) will both appear in neurons of the hippocampus before appearing in neurons of the isocortex. The proposed work seeks to bring the NIH's NIA one step closer to its goal of understand[ing] the nature of aging & the aging process, & diseases & conditions associated with growing older as it relates to AD, a ruthless, currently unstoppable, worldwide epidemic.